Methods of making and using live attenuated viruses

ABSTRACT

This disclosure provides a platform for making live, attenuated viruses. This disclosure also provides methods of using the live, attenuated viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e)to U.S. Application No. 62/222,322 filed Sep. 23, 2016.

TECHNICAL FIELD

This disclosure generally relates to live attenuated viruses andmaterials and methods for making such live attenuated viruses.

BACKGROUND

An attenuated vaccine is a live vaccine, which can be contrasted with akilled vaccine. An attenuated vaccine is created by reducing thevirulence of a pathogen, or eliminating the virulence of a pathogenunder certain conditions. Live attenuated vaccines provide betterprotection to the host, but safety concerns have limited their useoutside of the human population. These concerns are obviated by thematerials and methods described herein.

SUMMARY

The methods described herein provide a new platform that includes a cellline deficient in one or more universally expressed miRNAs. The platformdescribed herein allows for the production of live, miRNA-attenuatedvaccines that can be safely used, for example, in mammalian and avianspecies.

In one aspect, a method of making a live, attenuated virus is provided.Such a method generally includes providing a modified virus, wherein thevirus has been modified to comprise a miRNA-recognition nucleic acidsequence; culturing the modified virus in a miRNA knock-out cell line,wherein the knock-out cell line comprises a mutation or a transgene thatresults in the absence of the miRNA that, when present, binds to themiRNA-recognition nucleic acid sequence; and collecting the culturedvirus, wherein the cultured virus is annotated when introduced into acell expressing the miRNA.

Representative miRNAs include, without limitation, miRNA-23, miRNA-24,miRNA-29, miRNA-103, and miRNA-107. Representative viruses include,without limitation, an Influenza B virus, respiratory syncytial virus(RSV), polio virus, West Nile virus, Chikungunya virus, Ebola virus,Lassa virus, Dengue virus, SARS coronavirus, and Middle East RespiratorySyndrome (MERS) coronavirus.

In some embodiments, the modified virus includes one miRNA-recognitionnucleic acid sequence. In some embodiments, the modified virus includesa plurality of miRNA-recognition nucleic acid sequences.

In some embodiments, the mutation is an insertion, a deletion, asubstitution, or a point mutation. In some embodiments, the transgeneencodes at least one inhibitory nucleic acid (e.g., an antisense RNA, aRNAi, or a siRNA).

In another aspect, a live, attenuated virus is provided. In oneembodiment, a live, attenuated virus made by the methods describedherein is provided. In one embodiment, a live, attenuated virus isprovided that includes a miRNA-recognition nucleic acid sequence in itsgenome.

In still another aspect, a method of vaccinating a subject is provided.Such a method generally includes inoculating the subject with a live,attenuated virus as described herein. Representative subjects include,without limitation, humans, birds, cows, pigs, ferrets, dogs, or cats.

In yet another aspect, an article of manufacture is provided thatincludes a live, attenuated virus as described herein. In someembodiments, the article of manufacture further includes a knock-outcell line as described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic showing one embodiment of a miRNA-mediatedvaccine platform as described herein.

FIG. 1B is a schematic showing one embodiment of vaccine generation asdescribed herein.

FIG. 1C is a schematic showing the attenuated viruses that are producedby the methods described herein.

FIG. 2A is a photograph of a gel showing microRNA-mediated attenuation.

FIG. 2B is a photograph of a gel showing miRNA-mediated attenuation.

FIG. 3A shows CRISPR/Cas-mediated deletion of miR-24 from both loci inchromosomes 1 and 20 in MDCK cells. PCR primers specific for each miR-24location were used and run on an agarose gel demonstrating deletion ofmiR-24.

FIG. 3B is a RNA Northern Blot analysis from cells in FIG. 3A thatdemonstrates loss of miR-24 RNA expression.

DETAILED DESCRIPTION

Vaccines that rely upon live attenuated viruses generally provide betterprotection to the host and usually do not require booster vaccinations,but safety concerns have limited their use, with the exception of a fewinstances. The approach described herein is unique in thatspecies-ubiquitous microRNAs can be eliminated from cell lines used togrow and rescue the virus, but the virus contains recognition sequencesfor miRNAs that are ubiquitously expressed in at-risk species (e.g., thespecies to be vaccinated). The safety concerns usually associated withthe use of live attenuated viruses as vaccines are obviated by themethods described herein because miRNAs are used that are universallyexpressed, which allows for the use of live attenuated vaccines inanimals (e.g., domestic poultry) that have previously been hampered bysafety concerns with respect to consuming those animals by humans.

Previous efforts to use miRNAs to generate live attenuated vaccines havetaken advantage of the natural disparity in expression that some miRNAsexhibit within vaccine production systems (e.g., chicken eggs orMadin-Darby Canine Kidney (MDCK) epithelial cells) and the desiredvaccinated population (e.g., humans). These previous approaches havesignificant limitations, as there are no miRNAs that are absent from,for example, MDCK cells lines but present at levels abundant enough inhumans, at-risk mammals (e.g., canines, swine, felines, cattle), anddomesticated avian species to repress, or attenuate, virus replication.

Methods are described herein that allow for recognition sequences forspecific miRNAs to be engineered into the genomes of viruses, which canbe used to restrict its replication in the presence of the cognatemiRNA. FIG. 1 is a schematic showing one embodiment of a miRNA-mediatedvaccine platform described herein. FIG. 1A shows that broadly expressedmiRNAs can be knocked-out in a virus' host cell, and FIG. 1B is aschematic showing the subsequent generation of live, attenuated viruses,which can be used as vaccines. Specifically, FIG. 1B shows that a miRNArecognition sequence can be incorporated into the virus, and, using themiRNA-mediated vaccine platform shown in FIG. 1A, viral vaccines can berescued and amplified. As shown in FIG. 1C, the resulting live vaccinesare broadly attenuated in, for example, avian and mammalian speciesincluding humans.

The methods described herein are useful because they allow forgenerating live, attenuated viruses that can be used as vaccines inavian or mammalian species without the risk of spread into zoonotichosts. For example, vaccination of turkeys or chickens with a live,attenuated virus as described herein will not result in infections ofhumans. In addition, since multiple segments of a virus can be targetedsimultaneously, the possibility of reassortment of portions of thegenome, which is a critical risk of current live, attenuated vaccinemodalities, can be significantly diminished and even prevented by usingthe methods described herein.

miRNA Knockout Cell Line

A miRNA-mediated vaccine platform as described herein requires a cellline that has been engineered to be deficient in one or more miRNAs(e.g., a miRNA knock-out cell line). Such a cell line must be capable ofsupporting the life cycle of a virus. To date, it has not been possibleto generate a miRNA-targeted virus vaccine that is attenuated in bothmammalian and avian species; the platform described herein allows forthe development of such vaccines.

MicroRNAs are known in the art and are small regulatory RNAs thatcontrol mRNA levels within the cell. MicroRNAs are highly evolutionarilyconserved within the animal kingdom. Representative miRNAs include,without limitation, miRNA-23, miRNA-24, miRNA-29, miRNA-103, and/ormiRNA-107. In the miRNA-mediated vaccine platform described herein, itis desired that miRNAs that are expressed, e.g., abundantly expressed,e.g., ubiquitously expressed, in mammalian and/or avian species

Knock-out cell lines (i.e., cell lines deficient for one or more miRNAs)can be made using materials and methods well known in the art such as,without limitation, nucleases (e.g., CRISPR, TALENs, megaTALs,meganucleases, zinc finger nucleases); antibodies (e.g., Fab, Fab2,chimeric, humanized); or ligands, proteins, drugs, chemicals, or smallmolecules that competitively bind one or more miRNAs, that downregulatemiRNA expression, that increase miRNA degradation, or that causeintracellular depletion (e.g., by secretion) of miRNA. See, for example,Sambrook et al., 1990, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.

A skilled artisan also would appreciate that knock-out cell lines can bemade, for example, using a transgene that encodes at least oneinhibitory nucleic acid. Inhibitory nucleic acids are known in the artand can correspond to the sequence of the miRNA (e.g., a sense strand)or can be complementary to the sequence of the miRNA (e.g., an antisensestrand). Inhibitory nucleic acids are known in the art and include, forexample, antisense, RNAi, and siRNA. See, for example, U.S. Pat. Nos.5,453,566; 6,107,094; 6,506,559; 7,056,704; and 7,078,196.

The use of inhibitory nucleic acids also is referred to as RNAinterference (RNAi) or post-transcriptional gene silencing (PTGS), whichdescribes a biological process in which RNA molecules inhibit geneexpression, typically by causing the destruction of specific mRNAmolecules. Without being bound by theory, it appears that, in thepresence of an antisense RNA molecule that is complementary to anexpressed message (i.e., a mRNA), the two strands anneal to generatelong double-stranded RNA (dsRNA), which is digested into short (<30nucleotide) RNA duplexes, known as small interfering RNAs (siRNAs), byan enzyme known as Dicer. A complex of proteins known as the RNA InducedSilencing Complex (RISC) then unwinds siRNAs, and uses one strand toidentify and thereby anneal to other copies of the original mRNA. RISCcleaves the mRNA within the complementary sequence, leaving the mRNAsusceptible to further degradation by exonucleases, which effectivelysilences expression of the encoding gene.

Several methods have been developed that take advantage of theendogenous machinery to suppress the expression of a specific targetgene and a number of companies offer RNAi design and synthesis services(e.g., Life Technologies, Applied Biosystems). The use of RNAi caninvolve the introduction of long dsRNA (e.g., greater than 50 bps) orsiRNAs (e.g., 12 to 23 bps) that have complementarity to the targetgene, both of which can be processed by endogenous machinery.Alternatively, the use of RNAi can involve the introduction of a smallhairpin RNA (shRNA); shRNA is a nucleic acid that includes the sequenceof the two desired siRNA strands, sense and antisense, on a singlestrand, connected by a “loop” or “spacer” nucleic acid. When the shRNAis transcribed, the two complementary portions anneal intra-molecularlyto form a “hairpin,” which is recognized and processed by the endogenousmachinery.

It would be appreciated that a cell line as described herein can be madedeficient for more than one miRNA. For example, in some embodiments, acell line can be made deficient for at least two or more miRNAs usingmutagenesis and/or one or more transgenes. For example, one or moretransgenes can be introduced into a cell that encode one or moreinhibitory nucleic acids directed toward the same or different miRNAs.

Viruses Modified to Include a miRNA-Recognition Sequence

A miRNA-mediated vaccine platform as described herein also requires amodified virus. A modified virus as described herein includes at leastone nucleic acid sequence in its genome that is recognized by at leastone miRNA (referred to herein as a “miRNA-recognition nucleic acidsequence,” sometimes referred to as a “miRNA target sequence”). Modifiedviruses as described herein can be made using materials and methods thatare well known and routine in the art.

miRNA-recognition sequences would be understood to be a nucleic acidsequence, usually associated with a protein-encoding gene, to which amiRNA nucleic acid binds and directs their post-transcriptionalrepression. Therefore, a miRNA-recognition nucleic acid sequencetypically is complementary to at least a portion of the mature strand ofthe miRNA (e.g., the strand that is loaded into the RNA-inducedsilencing complex). Bartel (2009, Cell, 136(2):215-33), incorporated byreference in its entirety, provides a detailed description ofmiRNA-recognition sequences and how they can be identified.

As with the knock-out cell lines, it would be appreciated that amodified virus can contain one miRNA-recognition nucleic acid sequenceor a plurality of miRNA-recognition nucleic acid sequences. A pluralityof miRNA-recognition nucleic acid sequence can include two, three, four,or more miRNA-recognition nucleic acid sequences. A plurality ofmiRNA-recognition sequences can be the same or different recognitionsequences for the same miRNA and/or a plurality of miRNA-recognitionsequences can be recognition sequences for a plurality of miRNAs.

Nucleic Acids

Unless otherwise specified, nucleic acids referred to herein can referto DNA and RNA, and also can refer to nucleic acids that contain one ormore nucleotide analogs or backbone modifications. Nucleic acids can besingle stranded or double stranded, and linear or circular, both ofwhich usually depend upon the intended use.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

Nucleic acids can be isolated using techniques well known in the art.For example, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

It would be appreciated by the skilled artisan that complementary canrefer to, for example, 100% sequence identity between the two nucleicacids. In addition, however, it also would be appreciated by the skilledartisan that complementary can refer to, for example, slightly less than100% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99%sequence identity). In calculating percent sequence identity, twonucleic acids are aligned and the number of identical matches ofnucleotides between the two nucleic acids is determined. The number ofidentical matches is divided by the length of the aligned region (i.e.,the number of aligned nucleotides) and multiplied by 100 to arrive at apercent sequence identity value. It will be appreciated that the lengthof the aligned region can be a portion of one or both nucleic acids upto the full-length size of the shortest nucleic acid. It also will beappreciated that a single nucleic acid can align with more than oneother nucleic acid and hence, can have different percent sequenceidentity values over each aligned region.

The alignment of two or more nucleic acids to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid sequences tobe carried out across their entire length (global alignment). Chenna etal., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates thebest match between a query and one or more subject nucleic acidsequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more nucleotides can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the default parameters can be used (i.e., word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5); for an alignment of multiple nucleic acidsequences, the following parameters can be used: gap opening penalty:10.0; gap extension penalty: 5.0; and weight transitions: yes. ClustalWcan be run, for example, at the Baylor College of Medicine SearchLauncher website or at the European Bioinformatics Institute website onthe World Wide Web.

The skilled artisan also would appreciate that complementary can bedependent upon, for example, the conditions under which two nucleicacids hybridize. Hybridization between nucleic acids is discussed indetail in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and 11.45-11.57). Sambrook etal. disclose suitable Southern blot conditions for oligonucleotideprobes less than about 100 nucleotides (Sections 11.45-11.46). The Tmbetween a nucleic acid that is less than 100 nucleotides in length and asecond nucleic acid can be calculated using the formula provided inSection 11.46. Sambrook et al. additionally disclose Southern blotconditions for oligonucleotide probes greater than about 100 nucleotides(see Sections 9.47-9.54). The Tm between a nucleic acid greater than 100nucleotides in length and a second nucleic acid can be calculated usingthe formula provided in Sections 9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2×SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane. A nucleic acid molecule is deemed to hybridize to anucleic acid, but not to another nucleic acid, if hybridization to anucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater thanhybridization to another nucleic acid. The amount of hybridization canbe quantified directly on a membrane or from an autoradiograph using,for example, a PhosphorImager or a Densitometer (Molecular Dynamics,Sunnyvale, Calif.).

Methods of Making and Using a Live, Attenuated Virus

The cell lines described herein that are deficient in one or more miRNAscan be infected (e.g., transfected) with a modified virus as describedherein and used to make live, attenuated viruses, which can be used asvaccines. The relationship that is required between the deficientmiRNA(s) in the cell line and the miRNA-recognition nucleic acidsequence would be appreciated by a skilled artisan. That is, themiRNA(s) that are deficient in the knock-out cell line would, in theabsence of the deficiency (e.g., in the absence of a mutation(s) or atransgene(s)), recognize the miRNA-recognition nucleic acid sequencethat is contained within the modified virus.

The virus cultured can be collected and purified. Viruses can becollected and purified using any number of means and typically includesat least one cell culturing step in a suitable host cell or organism.See, for example, Acheson, 2011, Fundamentals of Molecular Virology,2^(nd) Ed., Wiley & Sons.

A live, attenuated virus vaccine made by the methods described hereincan be used to vaccinate a subject. The vaccination of a subject isroutine in the art and typically includes inoculating the subject withthe vaccine. Inoculation can be orally, rectally, topically, nasally,ocularly, intestinally, parenterally, or via the pulmonary tract. Routesof parenteral inoculation include intravenous, intramuscular,intradermal and subcutaneous administration. It would be appreciatedthat the live virus vaccine as described herein is attenuated in cellsexpressing the miRNA(s) (i.e., cells in the subject).

The methods described herein can be used as a platform to generate safeand effective vaccines in any number of subjects. For example, subjectscan include mammals (e.g., humans, cattle, swine, ferrets, canines andfelines) and avian species (e.g., domestic poultry species such aschickens, turkeys, and ducks).

The platform described herein can be used to produce live, attenuatedvirus vaccines using virtually any virus. The viruses that can beattenuated using the methods described herein include, withoutlimitation, RNA and DNA viruses, and single-stranded and double-strandedviruses. Non-limiting examples of viruses that can be attenuated usingthe methods described herein include influenza virus (e.g., Influenza Bvirus; e.g., H1N1, H2N2, H3N2, H5N1, H5N2, H7N9, and H9N9), respiratorysyncytial virus (RSV), polio virus, West Nile virus, Chikungunya virus,Ebola virus, Lassa virus, Dengue virus, SARS coronavirus, and MiddleEast Respiratory Syndrome (MERS) coronavirus.

It would be appreciated by a skilled artisan that the cell line that ismade deficient for one or more miRNAs is limited only by thecorresponding virus. That is, the cell line that is made deficient forone or more miRNAs needs to support the complete life cycle of the virusand needs to be able to produce new virions. Cell lines as used hereincan be, for example, human pulmonary epithelial cells (A549), caninekidney cells (MDCK), or African green monkey kidney cells (Vero).

Articles of Manufacture

This disclosure also provides for articles of manufacture (e.g., “kits”)that contain a live, attenuated virus as described herein. An article ofmanufacture also can include a corresponding knock-out cell line (e.g.,in culture, lyophilized). Additionally, an article of manufacture mayfurther include one or more buffers, adjuvants, or co-factors.

In some embodiments, an article of manufacture can include one or moresyringes for delivering a live, attenuated virus as described herein toan individual (e.g., to vaccinate an individual). In some embodiments, alive, attenuated virus can be provided (e.g., packaged) within one ormore syringes.

The components of an article of manufacture can be packaged togetherwith suitable packaging materials. Articles of manufacture also cancontain a package insert or package label having instructions thereonfor using the live, attenuated virus and/or the knock-out cell line.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Small RNA Deep Sequencing

microRNAs (miRNAs) were sequenced on the Illumina platform as previouslydescribed (Shapiro et al., 2010, RNA 16:2068-74; Pfeffer et al., 2005,Nat Methods, 2:269-76; and Langlois et al., 2013, Nature Biotech.,31:844-7). Briefly, RNA was extracted from the indicated tissue usingstandard TRIZOL protocols. RNA was run on a 15% denaturing Tris-Urea gelflanked by radiolabeled Decade markers (Ambion). Small RNA speciesbetween 15 and 30 nucleotides then were isolated, and the 3′ end of thesmall RNA fraction was ligated to an adapter using the Rnl2 Air™ Ligase(BIOO Scientific). The resulting ligated RNA then was separated from theunligated adapters by gel purification using size to discriminate. The5′ end then was ligated to an adapter RNA oligonucleotide using T4 RNAligase (NEB).

Following gel isolation, the ligation product was reverse transcribed,PCR amplified (21 cycles) and purified by agarose-based gelelectrophoresis. Quality of the small RNA library was assessed on theAgilent 2100 Bioanalyzer (Agilent). RNA libraries were sequenced on theIllumina Platform and mapped to pre-miRNAs as annotated on miRBase(mirbase.org on the World Wide Web). Percent of total was calculated bydividing the indicated miRNA species by the total number of miRBasemapped small RNAs in the library. Families of miRNAs were pooled (e.g.,miR-29a, miR-29b-1, miR-29b-2 and miR-29c-1 and miR-29c-2 were combinedand designated “miR-29”), since there is a high level of conservationamongst the mature miRNAs produced from families.

Table 1 shows the results from the experiments described herein as wellas data provided by Perez et al. (2009, Nature Biotechnol., 27:572-6),Langlois et al. (2012, Mol. Therapy, 20:367-75), Langlois et al. (2012,PNAS, 109:12117-22), and Langlois et al. (2013, Nature Biotechnol.,31:844-7). Table 1 is a heat map showing the amount of various miRNAs (%of total) in humans (A549 cells), ferret (respiratory tract; combineddata for nasal, trachea, bronchus and lung parenchyma), canine (MDCKcells), mouse (embryonic fibroblasts), and chicken (embryonic tissue).Table 1 shows that, while there is some heterogeneity in miRNAexpression across species, there are several miRNAs that are highlyexpressed across both species and cell types. Importantly, these miRNAsare expressed in species that are susceptible to influenza virusinfection.

TABLE 1 Percent of miRNA Expression Human Ferret Canine Mouse ChickenmiR-21 43.0 2.9 38.8 10.3 2.8 miR-24 6 3.2 3.8 3.2 11.6 miR-23 3.1 3.02.5 0.6 3.1 miR-103 1.4 0.3 3.2 1.3 3.1 miR-29 6.0 1.6 4.6 6.9 1.0miR-31 6.0 0 5.2 5.0 0.1 miR-125 1.8 3.8 1.7 0.7 0

Example 2 Western Blotting

The indicated MDCK cells were infected with wild type control ortargeted influenza viruses at a multiplicity of infection of one. 24hours post-infection, protein was harvested using a NP40 lysis bufferand run on a 4-15% gradient gel (BioRad). Protein was transferred tonitrocellulose blocked in 5% milk and probed using anti mouse NPantibody (NR43899 Bei resources) or anti sera from H7 HA immunized mice(gift from Dr. Peter Palese and Dr. Rong Hai, MSSM). Actin (anti mousePan Actin; Neomarkers) was used as a loading control. Protein was thenrevealed using anti mouse secondary antibodies conjugated to HRP(Roche).

The Western blots are shown in FIG. 2. MDCK cells lacking miR-126 (FIG.2A) and miR-151 (FIG. 2B) were engineered to express these miRNAs.Influenza viruses were then generated with miR-126 recognition sites(CGC AUU AUU ACU CAC GGU ACG A (SEQ ID NO:1)) incorporated into NP (FIG.2A) or miR-151 recognition sites (ACU AGA CUG UGA GCU CCU CGA (SEQ IDNO:2)) incorporated into H7 HA (FIG. 2B) (see Example 3 below). Wildtype MDCK or miRNA-expressing MDCK cells were then infected and probedfor targeted protein production. FIGS. 2A and 2B show that insertion ofmiRNA recognition sites in either the NP or the HA gene resulted inattenuated virus replication in the presence of the cognate miRNA butnot in the absence of the cognate miRNA.

Example 3 Generation of Recombinant miRNA-Targeted Influenza Viruses

miRNA-targeted recombinant influenza viruses were generated using theeight plasmid standard reverse genetics system (Fodor et al., 1999, J.Virol., 73:9679-82; and Langlois et al., 2013, Nature Biotech.,31:844-7). Four perfectly complementary recognition sites were clonedusing overlapping PCR or synthesized by Genewiz. To allow for insertioninto the influenza genome without disrupting the coding sequence of theprotein or the packaging signals of the viral RNA, the completepackaging signal 200 base pairs from the 5′ end of the vRNA wasduplicated and added at the end of the stop codon. A unique restrictionsite was added, allowing for insertion of the targeting sequence usinginfusion cloning systems (Clontech). The targeted plasmid was then usedwith 7 plasmids from unmanipulated segments to rescue virus in 293cells. Virus was then plaque purified and amplified in 10-day oldembryonated chicken eggs.

Example 4 Generation of miRNA Knock-Out Cells

miRNA knockout cells are generated by designing and transfecting guideRNAs flanking the 5′ and 3′ ends of the primary miRNA in the genome.Cells are co-transfected with a plasmid expressing the nuclease as wellas the cognate miRNA-targeted virus. Cells are clonally selected and theloss of miRNA locus is confirmed by PCR and small RNA Northern blotanalysis. MicroRNA targeted virus is inserted after the stop codon andupstream of a complete packaging signal. These viruses then are rescuedand amplified in the miRNA knockout cell lines.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A method of making a live, attenuated virus,comprising: providing a modified virus, wherein the virus has beenmodified to comprise a miRNA-recognition nucleic acid sequence;culturing the modified virus in a miRNA knock-out cell line, wherein theknock-out cell line comprises a mutation or a transgene that results inthe absence of the miRNA that, when present, binds to themiRNA-recognition nucleic acid sequence; and collecting the culturedvirus, wherein the cultured virus is annotated when introduced into acell expressing the miRNA.
 2. The method of claim 1, wherein the miRNAis selected from the group consisting of miRNA-23, miRNA-24, miRNA-29,miRNA-103, and miRNA-107.
 3. The method of claim 1, wherein the modifiedvirus comprises one miRNA-recognition nucleic acid sequence.
 4. Themethod of claim 1, wherein the modified virus comprises a plurality ofmiRNA-recognition nucleic acid sequences.
 5. The method of claim 1,wherein the virus is an Influenza B virus, respiratory syncytial virus(RSV), polio virus, West Nile virus, Chikungunya virus, Ebola virus,Lassa virus, Dengue virus, SARS coronavirus, and Middle East RespiratorySyndrome (MERS) coronavirus.
 6. The method of claim 1, wherein themutation is an insertion, a deletion, a substitution, or a pointmutation.
 7. The method of claim 1, wherein the transgene encodes atleast one inhibitory nucleic acid.
 8. The method of claim 7, wherein theat least one inhibitory nucleic acid is an antisense RNA, a RNAi, or asiRNA.
 9. A live, attenuated virus made by the method of claim
 1. 10. Amethod of vaccinating a subject, comprising inoculating the subject withthe live, attenuated virus of claim
 9. 11. The method of claim 10,wherein the subject is a human, a bird, a cow, a pig, a ferret, a dog,or a cat.
 12. An article of manufacture comprising the live, attenuatedvirus of claim
 1. 13. The article of manufacture of claim 12, furthercomprising a knock-out cell line.